Method for analyzing tumor aggressivity comprising measurement of polymerized actin

ABSTRACT

The invention concerns a method for analyzing tumor agressivity of cancer cells comprising measurement of the amount of stationary polymerized actin in a lysate of said cells. Advantageously, the measurement of the amount of stationary actin is carried out by fluorescence static polarization.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage entry of International ApplicationNumber PCT/FR2003/003802, filed Dec. 18, 2003. The disclosure of theprior application is hereby incorporated herein in its entirety byreference.

The objective of the present invention is a diagnostic or predictivetest in the cancer field. More precisely, the invention concerns amethod based on the direct measurement of the quantity of polymerizedactin in the steady state in a non-purified cellular lysate, as anindicator of both tumor aggressivity and sensitivity to anti-tumortreatment.

Based on one preferred form of implementation, the invention method isremarkable in that, apart from the cellular extract and the fluorescentactin, no supplementary purified protein addition is necessary tosimultaneously reveal discrimination between sensitive or resistantcells and cells that are or are not potentially aggressive.

The actin cytoskeleton is a protein structure that is essential tocellular survival. Notably, it enables the cell to maintain its shapeand its binding ability, to migrate, to communicate with adjacent cells.The cytoskeleton is an extremely dynamic structure, in a perpetual stateof alteration.

The dynamic properties of the cytoskeleton enable the cell tocommunicate with the external environment and to migrate to formmetastases, for example. These two properties are at the origin oftissue cohesion.

The dynamic properties of the cytoskeleton make its study extremelydifficult.

Many mechanisms for the regulation of actin polymerization have beendescribed in earlier work, among which can be cited mechanisms dependenteither on the Arp2/3 complex, or by the intervention of the family ofproteins bound to Ena/VASP.

Thus, the Arp2/3 complex, consisting of at least seven proteinsub-units, is regulated by the members of the Wiskott-Aldrich (WASP)syndrome family of proteins to activate the nucleation of theY-structured actin filaments (Machesky, Mullins et al. 1999).

For Ena/VASP, this is the family of proteins whose prototype is zyxin,among which can be mentioned LPP (LIM-containing lipoma preferredpartner), and TRIPE (thyroid receptor-interacting protein-6),characterized by the fact that they possess a rich proline domainfollowed by three LIM domains. The proteins belonging to this familyinteract with the proteins from the Ena/VASP family among which can bementioned VASP (Vasodilator stimulated phosphoprotein), Ena (with thedrosophila), Mena (equivalent of the Ena protein in mammals) and Ev1.Although these latter two families of proteins are clearly involved inthe regulation of this mechanism of actin polymerization, the sequenceof events responsible is not clearly explained (Fradelizi, Noireaux etal. 2001).

It seems that a single cell can contain several systems for actinpolymerization, each being responsible for a particular type of actinstructure.

The dynamic properties of the cytoskeleton render its analysis extremelycomplex, taking into account the different protein systems involved inthe process of actin polymerization and de-polymerization. If thebinding and resistance properties are related (Damian, Hazlehurst et al.2001; called Faute, Laurend et al. 2002), the invasive properties ofcancerous cells differ according to their resistance to anti-cancertreatments (Lopes, Ernst et al. 2002). In vivo, the resistant cells seemto have a greater invasive power (Mitsumoto, Kamura et al. 1998).

Thus, the study of only one of the two mechanisms in a purified systemin vitro may be incomplete for judging the cellular potential of actinpolymerization of an extract from a cell line or from a biologicalsample.

The research work carried out within the framework of the presentinvention has now made evident a net correlation between the measurementof the quantity of polymerized actin in the steady state on the onehand, and tumor aggressivity on the other.

The present invention thus has the objective of offering a simple andeffective diagnostic or predictive method for tumor aggressivity and thesensitivity of a subject to anti-cancer treatment. This objective isattained by means of a method which consists of measuring the quantityof polymerized actin in the steady state in a cellular extract from asample taken from a subject.

More specifically, the invention has the objective of creating a methodof analysis of tumor aggressivity in cancerous cells from a subjectconsisting of the measurement of the quantity of polymerized actin inthe steady state in a cellular extract from the subject.

Advantageously, the cellular extract is a lysate of the said cancerouscells.

Tumor aggressivity is understood to mean the invasive character of acancer or a cancerous cell line, that is both its metastatic potentialas well as the rapidity with which a primary tumor develops and grows.Tumor aggressivity is also understood to mean oncogenicity, that is, thecapacity of a cell line to result more or less efficiently in theappearance of a tumor after subcutaneous injection into a murine modellikely to receive this line. Tumor aggressivity is also understood tomean the absence of sensitivity to anti-cancer treatment.

The measurement taken on the cellular extract from the subject iscompared to one or more specific reference values of tissue analyzed inthe case of biological samples, or phenotype specifics, in the case ofcell lines.

The quantity of polymerized actin corresponds to the sum of all theF-form actin. This sum depends on the total quantity of actin,regardless of its form, form F or form G, but above all, on the whole ofthe mechanisms for regulating polymerization and de-polymerization.

Actin F is understood to mean the more or less long polymers of globularactin (actin G). The formation of actin F is a dynamic phenomenon, veryprecisely regulated by several different mechanisms. Actin F may sufferin a concomitant manner a phenomenon of polymerization of globular actinat one extremity of the filament and a phenomenon of depolymerization atthe other extremity.

An objective of the present invention is to be freed of the variouspaths of the regulation of actin polymerization by utilizing anintegrated system.

This objective is attained according to the invention by means of ameasurement of the quantity of polymerized actin in the steady statewhich takes into account the results of all the mechanisms forregulating the polymerization and de-polymerization of the actinfilaments, and more precisely, both the mechanisms of stimulation aswell as the mechanisms of inhibition of polymerization andde-polymerization. The steady state results from the equilibrium amongall these mechanisms for regulating actin polymerization at oneextremity of the actin filament and depolymerization at the otherextremity.

In an advantageous manner, the measurement of polymerized actin in thesteady state does not necessitate a measurement of an expression of aprotein (for example, zyxin or one of the proteins of the Arp2/3 complexor one of the proteins from the Ena/VASP family) and does notnecessitate the protein purification step or the addition of solidreactants (balls, for example).

The present invention enables a measurement of the quantity ofpolymerized actin in the steady state for a non-purified cellularextract, where all the activation/inhibition paths for the actinpolymerization and de-polymerization processes are integrated.

Thus, the quantity of polymerized actin in the steady state isunderstood to mean the quantity of polymerized actin when equilibriumbetween the actin polymerization at one extremity of the filaments andthe depolymerization at the other extremity is attained. As indicatedpreviously, the steady state is the result of all the paths forregulating the actin polymerization.

The measurement of the quantity of actin in the steady state can beaccomplished by any technique known to a person skilled in the art, suchas, for example, the static fluorescence polarization technique, alsoknown as static fluorescence anisotropy.

Anisotropy and polarization are two values related mathematically, andthus easily interchanged. They describe the same phenomenon.Fluorescence polarization permits a study of the interactions betweenmolecules by measuring the changes in the size of fluorescent moleculesin solution. This measurement is correlated with the size of thefluorescent molecule or the fluorescent molecular complex. In this case,the fluorescent molecule is an actin monomer (or actin G) bound to afluorochrome, in the case of Alexa 488, which is incorporated in theactin filaments (actin F) in the course of the polymerization.

Thus, according to one highly preferred method of implementation, themeasurement of the quantity of actin in the steady state of theinvention's method of analysis is realized by static fluorescencepolarization in the presence of actin monomers bound to a fluorochrome,which are incorporated into the actin filaments (actin F) formed in thecourse of the endogenous actin polymerization of the lysate.

In this form of implementation, the actin monomers bound to afluorochrome are added to a cellular lysate in a ratio ranging between1/80^(th) and 1/1600^(th) in relation to the quantity of endogenousactin.

The result of the test is a plateau fluorescence anisotropy value(<<STAFI>>) corresponding to the quantity of polymerized actin in thesteady state and an apparent constant (K_(obs)) for actin polymerizationresulting from the progressive incorporation of marked actin monomersover time until attainment of the steady state, that is the plateau ofthe curve.

By use of the GraphPad Prism® version 3 Software (GraphPad Software,Inc., San Diego, Calif.), for biostatistics, curve fitting, andscientific graphing, the experimental data are used to generate a curveadjusted to a 1st order equation such that:Y=ΔmA max(1−e ^(−k·t)) where

Y=the anisotropy value measured at time t

ΔmA max=<<STAFI>>, the maximum ordinate value at equilibrium

K=the constant K_(obs)

t=the time in seconds.

To predict a level of tumor aggressivity and sensitivity to ananti-cancer treatment, these two values must be compared either tospecific reference values of the tissue analyzed in the case of thebiological samples, or specifics of the phenotype, in the case of celllines.

A sample from a very aggressive cancer specimen, that is, one presentingeither an invasive character or an oncogenic character or again, havinglost the characteristic of sensitivity to anti-cancer treatments, willpresent a STAFI and a K_(obs) which is lower than the reference value,that is, the value obtained from similar samples that are only slightlyor non-aggressive.

For example, the normal plateau value for fluorescence anisotropy(<<STAFI>>=delta mA max) for a low-invasive line of melanoma (B16F0) is47 mA (K_(obs)=0.07). A line derived from it, described moreover as veryinvasive (B16F10) (Nakamura, Yoshikawa et al. 2002), presents a STAFIvalue of 37 mA (K_(obs)=0.02), distinctly lower that the value of theB16F0s. The <<STAFI>> and K_(obs) values obtained from invasive celllysates are distinctly lower than the values of the reference line (FIG.3).

Another example is the comparison of the <<STAFI>> value of oncogeniclines with the value obtained from non-oncogenic parental cell linestaken as references. The <<STAFI>> value of oncogenic lines isdistinctly lower than the value of non-oncogenic reference lines.

In the example of lines derived from NIH 3T3, the <<STAFI>> of theoncogenic line (NIH 3T3 EF) is equal to 35 mA compared to thenon-oncogenic reference lines (NIH 3T3 and NIH 3T3 EF zyxin) for whichthe <<STAFI>> is equal to 65 and 57 mA respectively (FIG. 1).

The oncogenic line BAF3 bcr-abl and the non-oncogenic line BAF3 present<<STAFI>> values of 40 mA and 58 mA respectively. Repression of thefusion oncogenic expression, responsible for the oncogenicity of theBAF3 bcr-abl line, induces the restoration of the value of <<STAFI>> toa value close to the reference line <<STAFI>> i.e., 52 mA and 58 mArespectively (FIG. 2).

A last example is the comparison of the <<STAFI>> value of breast cancercell lines more or less sensitive to anti-cancer treatment. The<<STAFI>> values of the two resistant lines (MCF7-MDR and MCF7-dox) aredistinctly lower than the value of the sensitive line (MCF7) taken as areference, i.e., 35 mA and 52 mA versus 71 mA (FIG. 4).

A preferred example of implementation of the method according to theinvention includes the following steps:

the lysis of cancerous cells in non-denaturing conditions for theproteins and the elimination of cellular debris,

the total protein dosage of the lysate,

the addition of actin monomers bound to fluorochrome,

the addition of substances required for endogenous actin polymerizationand the protection of the lysate proteins,

the measurement of the quantity of polymerized actin in the steady statein the lysate.

The present invention also aims to offer a method of identification ofmolecules likely to present an anti-cancer activity. Such a methodconsists of implementing the method of analysis of tumor aggressivity inthe presence of a sufficient quantity of one or more molecules to betested, and the determination of the capacity of the said molecule torestore a quantity of polymerized actin in the steady statecorresponding that of non-aggressive cells.

The work carried out within the framework of the present inventionresulted in the identification of molecules capable of restoring the<<STAFI>> value of aggressive cells to the level of that ofnon-aggressive cells. These molecules are likely to present ananti-cancer activity.

For example, the Jasplakinolide, added to the lysate of aggressive cells(NIH 3T3 EF) right at the time of the test, enabled the restoration ofthe <<STAFI>> value of these cells to a value near that of the <<STAFI>>of nononcogenic reference cells (NIH 3T3) (FIG. 5). Table 1 belowsummarizes the restoration of the value of delta mA max (<<STAFI>>,corresponding to the quantity of polymerized actin in the steady state)of oncogenic cells (NIH 3T3 EF, marked EF) to the level of the value ofnon-oncogenic cells (NIH 3T3) by the addition of the jasplakinolide(marked jaspla).

TABLE 1 Cell lines ΔmA max NIH 3T3 EF 30 NIH 3T3 62 NIH 3T3 BF +Jasplakinolide 65

The invention again concerns the application of the method of analysisof tumor aggressivity previously described to:

the evaluation of the invasive character of the said cells;

the evaluation of the oncogenicity of the cells;

the prediction of the sensitivity of the said cells to an anti-cancertreatment; the anti-cancer treatment consists for example ofradiotherapy or chemotherapy.

Sensitivity to anti-cancer treatment is understood to mean both theabsence of resistance to medication based on the MDR system (multi-drugresistance) bound to pump mechanisms of the P-gp family of proteins, aswell as the capacity of cancerous cells to go into apoptosis. These twophenomena can be in response to an anti-cancer treatment consisting ofradiotherapy or chemotherapy.

The invention is also directed at a kit for a diagnostic or predictivetest of tumor aggressivity, and more especially, for the measurement ofthe quantity of polymerized actin in the steady state for the evaluationof tumor aggressivity in a biological sample.

Such a kit includes:

a cell re-suspension medium for the lysis of cells,

the substances necessary for endogenous actin polymerization and theprotection of the lysate proteins,

the actin monomers bound to a fluorochrome,

an actin polymerization solution,

a general actin solution,

possibly the extract of aggressive and non-aggressive reference cells.

Other advantages and characteristics of the invention will appear in theexamples which follow, in which reference will be made to the appendeddrawings where;

FIG. 1 illustrates the measurement of actin polymerization in the steadystate (<<STAFI>>=delta mA max) in murine parental member cell lines,both non-oncogenic (NIH 3T3 and NIH 3T3 BF zyxin) and oncogenic (NIH 3T3EF).

FIG. 2 illustrates actin polymerization in the steady state (<<STAFI>>delta mA max) in parental non-member cell lines (hematopoietic murinecells), non-tumor (BAF3 and BAF3 Bcr-Abl′) and tumor (BAF3 Bcr-Abl′).

FIG. 3 illustrates the measurement of actin polymerization in the steadystate (<<STAFI>>=delta mA max) in parental cell lines from breastcarcinoma, sensitive (MCF7) and resistant (MCF7-Doxorubicin andMCF7-MDR).

FIG. 4 illustrates the measurement of actin polymerization in the steadystate (<<STAFI>>=delta mA max) in parental cell lines melanoma, withmore or less metastatic potential (B16F0<B16F10).

FIG. 5 illustrates the identification of a molecule (the jasplakinolide)capable of restoring the quantity of actin in the steady state(<<STAFI>>=delta mA max) of an oncogenic line to the level of thequantity of a non-oncogenic line.

I. METHODS

The technique of fluorescence polarization, also called staticfluorescence anisotropy enables the obtaining of a value dependent onthe size of the fluorescent molecule and the number of fluorescentmolecular complexes. That is, by the addition of a small proportion offluorescent monomer actin in a cellular extract, these monomers will beincorporated in the actin filament during polymerization, which willresult in an increase in the anisotropy value up to an apparent plateaucorresponding to equilibrium between the polymerization andde-polymerization of the actin filaments containing the fluorescentmonomers. The level of this plateau, called <<STAFI>> reflects thequantity of F at the steady state, that is, the cell binding potential,and the speed of reaching the plateau depends on the rapidity with whichthese filaments are formed. These two parameters are the indices oftumor aggressivity.

Method of cell lysis: For cells in culture, the cells might have to betrypsinized before being washed (washing solution: 135 mM NaCl, 2.7 mMKCl, 11.9 nM NaHCO₃, 0.36 mM NaH₂PO₄, 2 mM MgCl₂, 0.2 mM EGTA, 5.5 mMglucose, 0.3% albumin, pH>6.5). The cells are placed in suspension in asonication solution (10 mM Tri-HCl, pH 7.5, 10 mM EGTA and 2 mmMgCl₂+Roche protease inhibitors) at a rate of 50.10⁶ cells/ml andsonicated on ice, at a rate of 10 runs of 10 seconds separated by30-second pauses. The lysate is centrifuged for 30 min at 8000 rpm at 4°C. and filtered at 0.45 μm. The concentration in total proteins of thelysate is measured by the Bradford method, to be adjusted to 2 mg/mlwith a sonication solution. 0.4 mM final of ATP and DTT are added toconstitute the cellular lysate to be tested.

The actin monomer solution bound to a fluorochrome is prepared in thefollowing manner: the Alexa 488 actin stock solution (Molecular Probes)(7.3 mg/ml) is diluted to 1:200th in a G solution (5 mM Tri pH 8.1, 0.2mM CaCl₂ 0.2 mM DTT, 0.2 mM ATP) supplemented with 10% sucrose andultracentrifuged at 35,000 rpm, 120 min, at 4° C. in order to eliminatepossible actin filaments. This solution of marked actin monomers isstored at −80° C. in aliquots.

At the time of the test, the solution of marked actin monomers isdiluted to 1/3 in G solution. The apparatus used is a Beacon® 2000Fluorescence Polarization System (Invitrogen Corporation, Carlsbad,Calif.) fluorescence polarization spectrometer. Introduce into theBeacon® tube 167 μl of solution G and 3 μl of marked actin monomersolution diluted beforehand. After stabilization of the actin monomeranisotropy value at about 110 mA, add 4 μl of polymerization solution(2.5 M KCl, 50 mM MgCl₂, 25 mM ATP) and 20 μl of the cellular extract tobe tested at 2 mg/ml. The fluorescence anisotropy value is recorded overa period of about 200 seconds. The data are processed with the GraphPadPrism® version 3.0 software (GraphPad Software, Inc., San Diego,Calif.), for biostatistics, curve fitting, and scientific graphing. Thefluorescence anisotropy value of the marked actin monomers alone (about110 mA) is removed from the values that follow.

The whole set of cell lines is cultivated at 37° C. in a humidatmosphere containing 5% CO₂. They are maintained in a DMEM or RPMI(Gibco) medium, supplemented with 10% newborn veal serum or fetal vealserum (Gibco) and antibiotics (penicillin at 100 UI/mL and streptomycinat 100 μg/m L).

The NIH-3T3 line is a line of non-oncogenic murine fibroblasts.

The NIH-3T3-EF line is a tumor line derived from the precedent andcontains a coding ADNc for the fusion oncogene EWS-FLI in its genome.The expression of this protein is selected with the help of 2.5 μg/mL ofpuromycin.

The NIH-3T3-EF-zyxin line is a line derived from the precedent, whichhas lost its oncogenic character following transformation by a codingADNc for the human zyxin protein. The expression of this protein isselected with the help of geneticin.

The BAF3 line is a pre-lymphocytic murine line. It is maintained in thepresence of IL3.

The BAF3 Bcr-Abl line is a line derived from the precedent containing acoding ADNc for the fusion oncogene Bcr-Abl, the expression of which canbe suppressed by doxicycline. When this line is cultivated in theabsence of doxicycline and IL3, the oncogene is expressed, and it isthen marked BAF3 Bcr-Abl. When this line is cultivated in the presenceof doxicycline and IL3, the fusion oncogene is no longer expressed, andit is marked BAF3 bcr-abl′-(Dugray, Geay et al. 2001).

The B16F0 line is a murine melanoma line with a weak metastaticpotential (Nakamura, Yoshikawa et al. 2002).

The B16F10 line is a line derived from the precedent having acquired astrong metastatic potential as a result of 10 successive selections in asyngeneic murine model of pulmonary metastases (Nakamura, Yoshikawa etal. 2002). The MCF7 line is a line of weakly oncogenic human mammarycarcinoma.

The MCF7-Dox line is a line derived from the precedent, renderedresistant to doxorubicin by the regular addition of 10 μM of doxorubicinin a culture medium.

The MCF7-MDR line is a line derived from the MCF7 line, containing anADNc coding for the P-gp, responsible for the loss of cancerous cellsensitivity to chemotherapy.

II—RESULTS

1) Correlation Between Oncogenicity of Murine Fibroblast Cell Lines andthe Measured Values of <<STAFI>> and K_(obs).

FIG. 1 represents the determination of the value of delta mA max(<<STAFI>> corresponding to the quantity of polymerized actin in thesteady state) in three binding murine parental cell lines, non-oncogenic(NIH 3T3 and NIH 3T3 EF zyxin) and oncogenic (NIH 3T3 EF). Table 2 belowsummarizes the kinetics of actin polymerization in the presence ofcellular extracts by static fluorescence polarization.

TABLE 2 Cell lines Delta mA max K_(obs) NIH 3T3 65 0.086 NIH 3T3 EF 350.04 NIH 3T3 zyxin 57 0.033

The quantity of polymerized actin in the steady state (<<STAFI>>:fluorescence anisotropy value at the plateau) of the oncogenic line (NIH3T3 EF) is compared to that of the non-oncogenic parental line (NIH 3T3)(FIG. 1). The delta mA max (STAFI) of the oncogenic line (NIH 3T3 EF) isdistinctly lower than the delta mA max of the non-oncogenic line (NIH3T3), i.e., 35 and 65 respectively.

The expression of the zyxin in the oncogenic line NIH 3T3 EF leads to animportant reduction in the oncogenicity of this line (NIH 3T3 EF zyxin).The reduction in the oncogenicity of this line is correlated with therestoration of the delta mA max to a value near that of the referenceline, in this case, the NIH 3T3 line, i.e., 57 and 65 respectively.

2) Correlation Between the Oncogenicity of Non-Binding Re-LymphocytaryMurine Cell Lines and the Measured Values of <<STAFI>> and K_(obs).

FIG. 2 represents the determination of the value of delta mA max(<<STAFI>>, corresponding to the quantity of polymerized actin in thesteady state) in different non-binding, non-oncogenic (parental BAF3,BAF3 Bcr-Abl′) and oncogenic (BAF3 Bcr-Abl′) cell lines. Table 3summarizes the kinetics of actin polymerization in the presence ofcellular extracts by static fluorescence polarization.

TABLE 3 Cell lines Delta mA max K_(obs) parental BAF3 58 0.12 BAF3Bcr-Abl 40 0.05 Bcr-Abl 52 0.101

The quantity of polymerized actin in the steady state (<<STAFI>>:fluorescence anisotropy value at the plateau, that is the value of deltamA maximum) of the oncogenic line BAF3 Bcr-Abl′ transformed by thefusion oncogene Bcr-Abl, is compared to that of the non-oncogenicparental line (BAF3) (FIG. 2) (Dugray, Geay et al. 2001). For theoncogenic line (BAF3 Bcr-Abl′), the delta mA max is distinctly lowerthan the delta mA max of the non-oncogenic line (BAF3), i.e., 40 and 58respectively.

The same observation is made for the speed of actin polymerization,represented by the constant K_(obs). For the oncogenic line (BAF3Bcr-Abl′), K_(obs) is distinctly lower than the K_(obs) of thenon-oncogenic line (BAF3), i.e., 0.05 and 0.12 respectively.

The repression of the oncogene fusion expression by the doxicycline inthe BAF3 Bcr-Abl line leads to the loss of the oncogenicity of this line(Dugray, Geay et al. 2001). The repression of the oncogene expressionrestores the delta mA max of this oncogenic line to a value close tothat of the non-oncogenic reference line, i.e., 52 and 58 respectively.

3) Correlation Between the Sensitivity to an Anti-Cancer Treatment ofLines of Human Mammary Carcinoma and the Measured Values of <<STAFI>>and K_(obs).

FIG. 3 represents the determination of the value of delta mA max(<<STAFI>>, corresponding to the quantity of polymerized actin in thesteady state) in two parental melanoma cell lines, with more or lessmetastatic potential (B16F0<B16F10). Table 4 below summarizes thekinetics of actin polymerization in the presence of cellular extracts bystatic fluorescence polarization.

TABLE 4 Cell lines Delta mA max K_(obs) B16F0 47 0.071 B16F10 37 0.028

The quantity of polymerized actin in the steady state (<<STAFI>>:fluorescence anisotropy value at the plateau, that is the value of deltamA maximum) of the MCF7-Dox line, resistant to doxorubicin, is comparedto that of the sensitive parental line (MCF7) (FIG. 3). For theresistant line (MCF7Dox), the delta mA max is distinctly lower than thedelta mA max of the sensitive line (MCF7), i.e., 52 mA and 71 mArespectively.

In addition, the value of delta mA max of the resistant line MCF7-MDR,transfected by the gene coding for P-gp, is also comparable to the valueof the sensitive line (MCF7). For the resistant line (MCF7-MDR), thedelta mA max is also very distinctly lower than the delta mA max of thesensitive line (MCF7), i.e., 35 and 71 respectively.

4) Correlation Between the Metastatic Potential of Murine Melanoma CellLines (B16F0 and B16F10 and the Measured Values of <<STAFI>> andK_(obs).

FIG. 4 represents the determination of the value of delta mA max(<<STAFI>>, corresponding to the quantity of polymerized actin in thesteady state) in different binding, sensitive (MCF7) and resistant (MCF7Dox and MCF7 MDR) cell lines to an anti-cancer treatment. Table 5 belowsummarizes the kinetics of actin polymerization in the presence ofcellular extracts by static fluorescence polarization.

TABLE 5 Cell lines Delta mA max K_(obs) MCF7 71 0.0034 MCF7-Doxorubicin52 0.0034 MCF7 MDR 35 0.071

The quantity of polymerized actin in the steady state (<<STAFI>>:fluorescence anisotropy value at the plateau, that is, the value ofdelta MA maximum) of the B16F10 line, selected for its metastaticpotential starting with the B16F0 line (Nakamura, Yoshikawa et al.2002), is compared to that of the less metastatic parental line (B16F0)(FIG. 4). For the most aggressive line (B16F10), the delta mA max isdistinctly lower than the delta mA max of the nononcogenic line B16F0),i.e., 37 and 47 respectively.

The same observation is made for the speed of actin polymerization,represented by the constant K_(obs). For the most aggressive line(B16F10), K_(obs) is distinctly lower than the K_(obs) of the lessaggressive line (B16F0), i.e., 0.028 and 0.071 respectively.

5) Identification of Jasplakinolide as a Molecule which Restores theActin Polymerization Potential at the Steady State.

FIG. 5 represents the restoration of the value of delta mA max(<<STAFI>>, corresponding to the quantity of polymerized actin in thesteady state) of oncogenic cells (NIH 3T3 EF, marked EF) to the level ofthe value of non-oncogenic cells (NIH 3T3) by the addition ofjasplakinolide (marked jaspla).

The quantity of polymerized actin in the steady state (<<STAFI>>:fluorescence anisotropy value at the plateau, that is, the value ofdelta mA maximum) of oncogenic cells (NIH 3T3 EF, marked EF) wasmeasured after the addition of 10 μM of jasplakinolide to thepolymerization medium. The delta mA max of tumor cells determined at aninitial value of 35 mA is restored by the addition of jasplakinolide toa value close to that of the non-oncogenic line (NIH 3T3), 66 mA and 65mA respectively.

The same observation is made for the speed of actin polymerization,represented by the constant. Jasplakinolide restores the K_(obs) to avalue equal to 0.04, near a value that is close to the value obtainedwith non-oncogenic cells, i.e., 0.15 and 0.086 respectively.

BIBLIOGRAPHIC REFERENCES

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1. Method of analysis of the tumor aggressivity of cancerous cellscomprising the real time measurement of the quantity of polymerizedactin in the steady state in a lysate of the said cells wherein the realtime measurement of the quantity of actin in the steady state is carriedout by static fluorescence polarization in the presence of actinmonomers bound to a fluorochrome, the monomers being incorporated intothe actin filaments (actin F) formed during the endogenous actinpolymerization of the lysate.
 2. Method according to claim 1, whereinthe measurement carried out on the lysate is compared to one or morereference values of the quantity of polymerized actin in the steadystate.
 3. Method according to claim 1, wherein the quantity ofpolymerized actin corresponds to the sum of all the F-form actin. 4.Method according to claim 1, wherein the actin monomers bound to afluorochrome are added to the cellular lysate in a proportion rangingbetween 1/80^(th) and 1/1600^(th) in relation to the quantity ofendogenous actin.
 5. Method according to claim 1, including the stepsof: lysing cancerous cells in non-denaturing conditions for theproteins, and the eliminating cellular debris, determining the totalamount of proteins in the lysate, adding actin monomers bound to afluorochrome, adding one or more substances to activate endogenous actinpolymerization and protect the lysate proteins, wherein said substancesare selected from the group consisting of the Arp2/3 complex and theEna/VASP family of proteins, and measuring the quantity of polymerizedactin in the steady state in the lysate.
 6. Method of identification ofmolecules likely to present an anti-cancer activity, comprisingimplementing a method according to one of claims 1-3 and 4-5 in thepresence of said molecule, and determining the capacity of said moleculeto restore a quantity of polymerized actin in the steady statecorresponding to that of non-aggressive cells is determined.
 7. A methodof evaluating cancer cells to determine their invasiveness, comprisingcarrying out the method according to one of claims 1-3 and 4-5.
 8. Amethod of evaluating cancer cells to determine their oncogenicity,comprising carrying out the method according to one of claims 1-3 and4-5.
 9. A method of evaluating cancer cells to determine theirsensitivity to an anti-cancer treatment, comprising carrying out themethod according to one of claims 1-3 and 4-5.
 10. The method accordingto claim 9, wherein the said anti-cancer treatment consists ofradiotherapy or chemotherapy.